The present invention relates to the use of morphinan derivatives as well as their bases or salts of physiologically compatible acids as regulators for the nociceptin/orphanin FQ ligand ORL1 receptor system and for the production of a medicament.
The heptadecapeptide nociceptin/orphanin FQ is an endogenous ligand of the ORL1 (opioid receptor-like) receptor (Meunier et al., Nature 377, 1995, pp. 532-535) that belongs to the family of opioid receptors and can be found in many regions of the brain and spinal cord (Mollereau et al., FEBS Letters, 341, 1994, pp. 33-38, Darland et al., Trends in Neurosciences, 21, 1998, pp. 215-221). The peptide is characterised by a high affinity, with a Kd value of around 56 pM (Ardati et al., Mol. Pharmacol. 51, pp. 816-824), and by a high selectivity for the ORL1 receptor. The ORL1 receptor is homologous to the xcexc, xcexa and xcex4 opioid receptors, and the amino acid sequence of the nociceptin/orphanin FQ peptide has a strong similarity to those of the known opioid peptides. The activation of the receptor induced by nociceptin/orphanin FQ leads via the coupling with Gi/o proteins to an inhibition of adenylate cyclase (Meunier et al., Nature 377, 1995, pp. 532-535). Also, at the cellular level there are functional similarities between the xcexc, xcexa and xcex4 opioid receptors and the ORL1 receptor as regards the activation of the potassium channel (Matthes et al., Mo. Pharmacol. 50, 1996, pp. 447-450; Vaughan et al., Br. J. Pharmacol. 117, 1996, pp. 1609-1611) and the inhibition of the L, N and P/Q type calcium channels (Conner et al., Br. J. Pharmacol. 118, 1996, pp. 205-207; Knoflach et al., J. Neuroscience 16, 1996, pp. 6657-6664).
The nociceptin/orphanin FQ peptide exhibits after intercerebroventricular application a pronociceptive and hyperalgesic activity in various animal models (Reinscheid et al., Science 270, 1995, pp. 792-794; Hara et al., Br. J. Pharmacol. 121, 1997, pp. 401-408). These results may be explained as inhibition of stress-induced analgesia (Mogil et al., Neurosci. Letters 214, 1996, pp. 131-134, as well as Neuroscience 75, 1996, pp. 333-337). In this connection an anxiolytic activity of the nociceptin/orphanin FQ peptide was also detected (Jenck et al., Proc. Natl. Acad. Sci. USA 94, 1997, 14854-14858).
On the other hand an antinociceptive effect of nociceptin/orphanin FQ, in particular after intrathecal application, was also found in various animal models. Nociceptin/orphanin FQ inhibits the activity of kainate-stimulated or glutamate-stimulated posterior root ganglioneurons (Shu et al., Neuropeptides, 32, 1998, 567-571) or glutamate-stimulated spinal cord neurons (Faber et al., Br. J. Pharmacol., 119, 1996, pp. 189-190); nociceptin/orphanin FQ has an anti- nociceptive action in the tail-flick test in mice (King et al., Neurosci. Lett., 223, 1997, 113-116), in the flexor-reflex model in rats (Xu et al., NeuroReport, 7, 1996, 2092-2094) and in the formalin test in rats (Yamamoto et al., Neuroscience, 81, 1997, pp. 249-254). An antinociceptive effect of nociceptin/orphanin FQ was also detected in neuropathic pain models (Yamamoto and Nozaki-Taguchi, Anesthesiology, 87, 1997), which is all the more interesting in that the efficacy of nociceptin/orphanin FQ increases after axotomy of spinal nerves. This is in contrast to conventional opioids, whose efficacy decreases under these conditions (Abdulla and Smith, J. Neurosci., 18, 1998, pp. 9685-9694).
The nociceptin/orphanin FQ ligand ORL1 receptor system is also involved in the regulation of further physiological and pathophysiological processes. These include, inter alia, learning and the formation of memory (Sandin et al., European. J. Neurosci., 9, 1997, pp. 194-197; Manabe et al., Nature, 394, 1997, pp. 577-581), auditory perception (Nishi et al., EMBO J., 16, 1997, pp. 1858-1864), food intake (Pomonis et al., NeuroReport, 8, 1996, pp. 369-371), blood pressure regulation (Gumusel et al., Life Sci., 60, 1997, pp. 141-145; Campion and Kadowitz, Biochem. Biophys. Res. Comm., 234, 1997, pp. 309-312), epilepsy (Gutierrez et al., Abstract 536.18, Society for Neuroscience, Vol. 24, 28th Ann. Meeting, Los Angeles, Nov. 7-12, 1998) and diuresis (Kapista et al., Life Sciences, 60, 1997, PL 15-21).
Morphinan derivatives as well as processes for their production are known from WO 98/22467, WO 95/31463 and WO 95/31464. These compounds are described as xcex4-selective opioid agonists and opioid antagonists for the treatment of conditions such as for example shock, constipation, mental disorders, eating disorders, injury to the central nervous system, alcoholism and immune function disorders.
The object of the present invention was to provide medicaments that act on the nociceptin/orphanin FQ ligand ORL1 receptor system and are thus suitable for treating neuropathic pain and/or anxiolysis and/or depression and/or diuresis and/or urinary incontinence and/or hypotension and or hypertension and/or senile dementia and/or Alzheimer""s disease and/or general cognitive dysfunctions and/or tinnitus and/or impaired hearing and/or epilepsy and/or obesity and/or cachexia.
It has now surprisingly been found that morphinan derivatives of the following general formula I exert an influence on the control of various physiological and pathophysiological processes in which the nociceptin/ orphanin FQ ligand ORL1 receptor system is involved. The aforementioned processes include, inter alia, the sensation of neuropathic pain, anxiety behavior, learning and memory formation, blood pressure regulation, hearing, food intake, epilepsy and diuresis.
The present invention accordingly provides for the use of morphinan derivatives of the general formula I 
wherein
R1 denotes H, a C1-4 alkyl radical or a C2-4 alkenyl radical,
R2 denotes H, a C1-18 alkyl radical, preferably a C1-10 alkyl radical, a C2-18 alkenyl radical, preferably a C2-10 alkenyl radical, a heterocyclyl radical or an aryl radical,
R3 denotes a C1-18 alkyl radical, preferably a C1-10 alkyl radical, a C2-18 alkenyl radical, preferably a C2-10 alkenyl radical, a C3-7 cycloalkyl, aryl or heterocyclyl radical bound via a C1-4 alkylene group, or a C3-7 cycloalkyl, aryl or heterocyclyl radical bound via a C2-4 alkenylene group,
R4 denotes H, a C1-18 alkyl radical, preferably a C1-10 alkyl radical, a C2-18 alkenyl radical, preferably a C2-10 alkenyl radical, a C3-7 cycloalkyl or aryl radical bound via a C1-4 alkylene group, or a C3-7 cycloalkyl or aryl radical bound via a C2-4 alkenylene group, and
X denotes O or NR5 where
R5 denotes H, a C1-18 alkyl radical, preferably a C1-10 alkyl radical, a C2-18 alkenyl radical, preferably a C2-10 alkenyl radical, a C3-7 cycloalkyl, aryl or heterocyclyl radical bound via a C1-4 alkylene group, or a C3-7 cycloalkyl, aryl or heterocyclyl radical bound via a C2-4 alkenylene group,
and/or their enantiomers, diastereomers, or physiologically compatible salts, as regulators for the nociceptin/orphanin FQ ligand ORL1 receptor system.
The term alkyl radicals also includes hydrocarbons at least singly substituted preferably by halogen, particularly preferably by fluorine. If these contain one or more substituents, then the latter may be identical or different. Preferably the alkyl radicals are methyl, ethyl, propyl, 1-methylethyl, butyl, 1-methylpropyl, 2-methylpropyl, 1,1-dimethylethyl, pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, hexyl, 1-methylpentyl, heptyl, nonyl or decanyl.
The term alkenyl radicals also includes hydrocarbons that contain at least one double bond, and may be at least singly substituted, preferably by halogen, particularly preferably by fluorine. If the alkenyl radical contains more than one substituent, then these may be identical or different. Preferably the alkenyl radicals are 2-propenyl, 2-butenyl, 1-methyl-2-propenyl or 2-methyl-2-propenyl.
The term aryl radical also includes phenyl or naphthyl radicals at least singly substituted by an OH, a halogen, preferably F and/or Cl, a CF3, a C1-6 alkyl, a C1-6 alkoxy, a C1-7 cycloalkoxy, a C3-7 cycloalkyl, a C2-6 alkylene or phenyl radical. The phenyl radicals may also be condensed with further rings.
The term heterocyclyl radical is understood to include saturated as well as unsaturated heterocyclic compounds, preferably 5-7-membered heterocyclic compounds that contain at least one heteroatom, preferably nitrogen, oxygen and/or sulfur, particularly preferably nitrogen and/or oxygen. Preferably the saturated heterocyclics are 1,4-dioxane, tetrahydrofuran or 1,4-thioxane. Preferably the unsaturated heterocyclics are furan, thiophene, pyridine, pyrimidine, thiazole, oxazole, isoxazole, pyridazine, pyrazine, quinoline, isoquinoline, phthalazine or quinazoline.
Preferred are morphinan derivatives of the general formula I in which R1 denotes H or a C1-4 alkyl radical; R2 denotes H or a C1-4 alkyl radical; R3 denotes a C1-4 alkyl radical or a C2-4 alkenyl radical; R4 denotes H, a C1-4 alkyl radical, a C3-7 cycloalkyl or aryl radical bound via a C1-4 alkylene group; and X denotes NR5 wherein R5 denotes a C1-10 alkyl, a C2-10 alkenyl, a C3-7 cycloalkyl or aryl radical bound via a C1-4 alkylene group, or a C3-7 cycloalkyl or aryl radical bound via a C2-4 alkenylene group.
Also preferred are morphinan derivatives of the general formula I in which R1 denotes H or a C1-4 alkyl radical; R2 denotes H; R3 denotes a C1-4 alkyl radical or a C2-4 alkenyl radical; R4 denotes H, a C1-4 alkyl radical or a C3-7 cycloalkyl or aryl radical bound via a C1-4 alkylene group; and X denotes NR5 wherein R5 denotes a C2-10 alkenyl radical, a C3-7 cycloalkyl or aryl radical bound via a C1-4 alkylene group, or an aryl radical bound via a C2-4 alkenylene group.
Particularly preferred are morphinan derivatives of the general formula I in which R1 denotes H, R2 denotes H, R3 denotes a C2-4 alkenyl radical, R4 denotes H and X denotes NR5, wherein R5 denotes an aryl radical bound via a C1-4 alkylene group or an aryl radical bound via a C2-4 alkenylene group.
Particularly preferred are the following morphinan derivatives:
17-allyl-6,7-didehydro-4,5xcex1-epoxy-3,14-dihydroxy-1xe2x80x2-(o-chlorobenzyl)indolo[6,7:2,xe2x80x23xe2x80x2]-morphinan hydrochloride,
17-allyl-6,7-didehydro-4,5xcex1-epoxy-3-hydroxy-14-(m-chloro-methoxyphenyl)1xe2x80x2-(m-chlorobenzyl)indolo[6,7:2,xe2x80x23xe2x80x2]-morphinan hydrochloride, or
17-cyclopropylmethyl-6,7-didehydro-4,5xcex1-epoxy-3-hydroxy-14-(o-chloromethoxyphenyl) 1xe2x80x2-(o-chlorobenzyl)indolo[6,7:2,xe2x80x23xe2x80x2]-morphinan hydrochloride
as ORL1 receptor antagonists, and
17-cyclopropylmethyl-6,7-didehydro-4,5xcex1-epoxy-3-hydroxy-14-(methoxynaphthalene)1xe2x80x2-(xcex2-methylnaphthalene)indolo-[6,7:2,xe2x80x23xe2x80x2]-morphinan hydrochloride or
17-cyclopropylmethyl-6,7-didehydro-4,5xcex1-epoxy-14-hydroxy-3-benzyloxy-1xe2x80x2-(p-methoxycarbonylmethylphenyl)-indolo[6,7:2,xe2x80x23xe2x80x2]-morphinan hydrochloride
as ORL1 receptor agonist.
The present invention also provides for a medicament and a method for treating neuropathic pain and/or anxiolysis and/or depression and/or diuresis and/or urinary incontinence and/or hypotension and or hypertension and/or senile dementia and/or Alzheimer""s disease and/or general cognitive dysfunctions and/or tinnitus and/or impaired hearing and/or epilepsy and/or obesity and/or cachexia using the morphinan derivatives of the general formula I as regulators for the nociceptin/orphanin FQ ligand ORL1 receptor system.
The present invention furthermore provides a method and a medicament for treating neuropathic pain and/or anxiolysis and/or depression and/or diuresis and/or urinary incontinence and/or hypotension and or hypertension and/or senile dementia and/or Alzheimer""s disease and/or general cognitive dysfunctions and/or tinnitus and/or impaired hearing and/or epilepsy and/or obesity and/or cachexia using the morphinan derivatives of the general formula I.
For the preparation of the corresponding pharmaceutical formulations, in addition to at least one morphinan derivative of the formula I there are also used carrier materials, or excipients, such as fillers, solvents, diluents, colourants and/or binders. The choice of the auxiliary substances as well as the amounts thereof to be used depends on whether the medicament, or pharmaceutical composition, is to be administered orally, intravenously, intraperitoneally, intradermally, intramuscularly, intranasally, buccally or topically. For oral application preparations in the form of tablets, sugar-coated pills, capsules, granules, drops, juices and syrups are suitable, while for parenteral, topical and inhalative administration, solutions, suspensions, easily reconstitutable dry preparations as well as sprays are suitable. Compounds of the formula I according to the invention in a depot form, in dissolved form or in a plaster, optionally with the addition of agents promoting penetration of the skin, are suitable percutaneous application preparations. Orally or percutaneously usable preparation forms can provide for the delayed release of the compounds of the formula I according to the invention.
The amount of active constituent to be administered to the patient varies depending on the patient""s weight, type of application, medical indications and severity of the condition. Normally 0.1 to 1 mg/kg body weight of at least one morphinan derivative of the formula I is administered.
The morphinan derivatives of the general formula I according to the invention were identified with a reporter gene system as ORL1 receptor agonists or as ORL1 receptor antagonists. This reporter gene system is based on the expression of the human ORL1 receptor in CHO-K1 cells, which carry a cAMP-sensitive luciferase gene (Wnendt et al., Regulatory Peptides, 80, 1999, p. 127 the entire disclosure of which is incorporated herein by reference). The formation of cAMP is induced by forskolin, an adenylate cyclase activator. Since the ORL1 receptor in the binding of the natural ligand nociceptin/orphanin FQ reduces the formation of cAMP by inhibiting adenylate cyclase, the effect of a substance as agonist or antagonist on the ORL receptor can be detected by a change in the cAMP-dependent luciferase formation.
ORL1 receptor agonists have the same effect as the natural ligand nociceptin/orphanin FQ. Also, they inhibit in a concentration-dependent manner luciferase formation due to the binding to the ORL1 receptor. The potency, expressed as the IC50 value, indicates the concentration at which the semi-maximum effect of the agonist is reached.
The ORL1 receptor antagonists compete with the natural ligand nociceptin/orphanin FQ for the binding to the ORL1 receptor. The inhibition of luciferase formation due to the agonists is lifted to the extent that the antagonist binds to the ORL1 receptor. The dose-effect curve of the natural ligand nociceptin/orphanin FQ is displaced to higher concentration ranges. The effect of the antagonists is described by the pKB value. This logarithmic quantity indicates the concentration at which the potency of the natural ligand nociceptin/orphanin FQ is reduced by a factor of 2 (Kenakin, T., Pharmacological Analysis of Drug-Receptor Interaction, 3rd Edition, Lippincott-Raven, 1997, Philadelphia, New York).
The reporter gene system was constructed as follows:
A cDNA coding for the ORL1 receptor was cloned using standard methods from THP-1 cells (European Cell Culture Collection, Porton Down, Great Britain) of a human monocyte cell line and integrated into the plasmid pZeoSV (Invitrogen, Leek, Netherlands). This plasmid, pZeoORL17, was used for the transfection of a cell line, CHO-K1/pSE66/K9, that contains the cAMP reporter plasmid pSE66. The plasmid pSE66 was constructed on the basis of the plasmid pMAMneo-LUC (Clontech, Palo Alto, Calif.) by replacing the RSV promoter of this plasmid by a promoter region in which six CRE elements (cAMP-responsive elements, Comb et al., Nature, 323, 1986, pp. 353-356; Montminy et al., Proc. Natl. Acad. Sci. USA, 83, 1986, pp. 6682-6686; Short et al., J. Biol. Chem., 261, 1986, pp. 9721-9726) lie upstream of a promoter. The SV40 promoter derives from the plasmid pGL2-promoter (Promega, Madison, Wis.). The promoter region constructed in this way controls in the plasmid pSE66 the expression of the luciferase gene from pMAMneo-LUC. Furthermore, pSE66 carries a G418 resistance gene under the control of a further SV40 promoter. The cell line CHO-K1 (European Cell Culture Collection, Porton Down, Great Britain) was rendered stable with pSE66 and the clone CHO-K1/pSE66/K9 was selected for the transfection with pZeoORL17. Monoclonal pZeoORL17-tranformants were selected in the nutrient mixture F-12 (Ham) with glutamine (Gibco-BRL, Weiterstadt, Germany), amplified with 50 xcexcg/ml of G418 (Gibco-BRL, Weiterstadt, Germany) and 200 xcexcg/ml of zeocin (Invitrogen, Leek, Netherldands).
Nociceptin/orphanin FQ sensitive clones were identified by incubating in each case 20,000 cells of a clone in a 96-well microtiter plate in a volume of 100 xcexcl for 6 hours with 1 xcexcM forskolin (RBI, Deisenhofen, Germany) in the presence or absence of 10 xcexcM of nociceptin/orphanin FQ, and were then measured using the luciferase detection kit (Roche, Mannheim, Germany) (Ford and Leach, Methods in Molecular Biology, 102, 1998, pp. 3-20).
The clone CHO-K1/pSE66/K9/pZeoORL17/K21 was selected for the analysis of test compounds, the test compounds being dissolved in dimethyl sulfoxide and the end concentration of dimethyl sulfoxide being set at 1% (v/v). The antagonist test was carried out in the presence of 10 nM of nociceptin/orphanin FQ.
Furthermore a receptor binding assay with 3H-nociceptin/ orphanin FQ was carried out with the compounds according to the invention using membranes of recombinant CHO-ORL1 cells. This test system was implemented according to the method proposed by Ardati et al. (Mol. Pharmacol., 51, 1997, pp. 816-824). The concentration of 3H-N/OFQ in these experiments was 0.5 nM. The binding assays were performed in each case with 20 xcexcg of membrane protein per 200 xcexcl of batch in 50 mM of Hepes, pH 7.4, 10 mM MgCl2 and 1 mM EDTA. The binding to the ORL1 receptor was determined using in each case 1 mg of WGA-SPA beads (Amersham-Pharmacia, Freiburg) by incubating the batch for one hour at room temperature followed by measurement in a Trilux scintillation counter (Wallac, Finland). The affinity is given as the Ki value.
The production of the morphinan derivatives of the general formula I is described in WO 95/31463 and WO 95/31464, the disclosures of which are incorporated herein by reference in their entirety.